Project Summary Novel cell therapies, including gene therapy for Sickle Cell Disease (SCD) require a relative large number of stem cells collected from peripheral blood for cell manipulation and subsequent transfusion. Current methods rely on centrifugal apheresis, which is associated with poor collection efficiency owing to the altered migration patterns of SCD red cells. Even when collected, products are highly contaminated with red cells requiring further processing and the corresponding cell loss alters transduction strategies and the net therapeutic dose. Poor collections limit the ability of SCD patients to fully participate in clinical cell therapy trials, and there is a great need to overcome this problem. SCD patients more likely require repeated collections using plerixafor alone, increasing costs and morbidity. To improve PBSC collections, we will test an alternative method of cell separation, acoustic apheresis, for the ability to further separate and enrich stem cells from peripheral blood to allow for the treatment of SCD patients. Current collections from SCD patients yield large volume products with high hematocrits and acoustophoresis can improve this obstacle. In the first aim of the proposal, we will test acoustophoresis on normal blood and apheresis products to understand how various peripheral blood cells can be fractionated and collected. We will then test acoustophoresis on sickle blood and characterize the functional dynamics of red cells following the separation procedure. These studies are geared toward the goal of successfully collecting and enriching stem cells from SCD patients, and will furthermore elucidate the heterogeneity of red cells and their functional dynamics in SCD with correlates for iron deficiency anemia and other hemoglobinopathies. In the second aim of the proposal, we will develop instrumentation to perform separations at higher flow rates, with the vision that acoustophoresis could in the future supplement conventional apheresis in real time as products are collected. This technology could safely collect both cellular and red cell fractions for clinical use. Acoustophoresis is best studied and introduced clinically by Transfusion Medicine as this is where patients intersect with those who collect cells and those who treat. As more trials of cell therapy launch, Transfusion Medicine has the ability to transform the field and directly aid patients to achieve therapy.